1. Field of the Invention
The present invention relates to a method for increasing a storage lipid content in a plant seed. The present invention also relates to a plant with an increased storage lipid content in its seeds. Further, the present invention relates to seeds with an increased storage lipid content.
2. Description of the Related Art
Plants belonging to the genus Brassica, which bear seeds containing lipid in an amount of about 40% by weight, are now cultivated as oil plants at many places in the world. As for rapeseed, a variety has been created by means of a cross breeding method in order to decrease the content of harmful substances such as erucic acid and glucosinolate. Until the present, a lipid content of about 40% based on the weight of seed has been achieved by means of cross breeding. However, the amount of vegetable oil consumption is increasing as compared with animal oil because people are interested in healthy life. Therefore, achievement of a high lipid content is ranked to be a first target in the breeding for rapeseed, and it is desired to further increase the lipid content.
However, the following problems have arisen. Namely, there is no variety to be used as a genetic source having a high lipid content for mating. The conventional breeding involves an extremely laborious and time-consuming process in which a species having an objective character is selected from random variations, which is non-oriented mutation so that a pure line is established. Therefore, the increase in lipid content achieved by the breeding based on mating has a certain limit in the present circumstances.
On the contrary, the process to create a recombinant plant based on the use of gene manipulation can provide a breeding method associated with extremely high orientation, because only a gene concerning an objective character is dealt with to make artificial modification and introduction. For example, the lipid composition in a plant has been modified by introducing antisense DNA, i.e., a DNA sequence coding for antisense RNA (antisense oligonucleotide). Those known for such modification include, for example, the fact that the oleic acid content is decreased and the stearic acid content is increased by introducing, into rapeseed, an antisense gene of stearate desaturase (Proc. National Acad. Sci. USA, 89, 2624, 1992).
Storage compounds in seeds are different in quantitative ratio depending on plants which accumulate the storage compounds. However, the storage compounds principally include lipid, protein, and carbohydrate. It is known that these substances are produced and accumulated in an approximately simultaneous period during the development period of seed, i.e., cell division and cell elongation stage, and their biosynthetic pathways are closely correlated with each other. Namely, an assimilated product produced by photosynthesis is used as a starting material to proceed biosynthesis of the respective substances. Lipid, protein, and carbohydrate are synthesized and accumulated as final products. Pyruvic acid is one of intermediate products of the biosynthesis of the storage compounds in seed. Pyruvic acid can be a substrate for synthesizing lipid, protein, and carbohydrate. In the case of rapeseed, lipid and protein are principally accumulated as storage compounds, and carbohydrate is scarcely accumulated. Accordingly, lipid synthesis is possibly affected if any one of reactions in the biosynthesis pathway for storage protein in seed is inhibited. Those conceived as a method to achieve such a situation include inhibition of biosynthesis of storage protein in seed based on the use of an antisense gene directed to pyruvate kinase which synthesizes pyruvic acid and ATP from phosphoenolpyruvate and ADP, and an antisense gene directed to phosphoenolpyruvate carboxylase which uses, as a substrate, phosphoenolpyruvate to serve as a substrate for pyruvate kinase.
In all eukaryotes, pyruvate kinase is found within the cytosol. However, in plants, pyruvate kinase is located also in plastids. These two types of pyruvate kinase differ significantly in their molecular and kinetic characteristics and are immunologically unrelated proteins. Among them, those determined and reported for the former include a complete nucleotide sequence of cDNA of castor oil seed (Plant Physiol., 96, 1238-1288, 1991) and a nucleotide sequence of a cDNA of a tobacco cDNA (Plant Mol. Biol., 27 (1) 79-89, 1995). Those determined and reported for the latter include a complete nucleotide sequence of potato cDNA and an amino acid sequence for castor pyruvate kinase (Plant Mol. Biol., 15, pp. 665-669, 1990), a nucleotide sequence of soybean cDNA (Plant Physiol., 102, 1345, 1993; it has been registered in Genbank under a number of L08632), and a part of a nucleotide sequence for rice pyruvate kinase (EMBL OSAD 11727). However, the relation between the genes and distribution of storage compounds has not been clarified. In addition, in the case of the use of transformation based on the antisense method, homology of the sequence is especially important. Accordingly, it is desirable to use a gene obtained from an objective to be utilized. It has been desired to isolate a gene of pyruvate kinase originating from an oil plant.